Summary: Oligodeoxyribonucleoside phosphorothioates (PS-ODNs) have been, and are still, extensively studied as potential therapeutic agents against various types of cancer and infectious diseases in humans. Given that these oligonucleotide analogues are P-chiral, each of the internucleotidic phosphorothioate linkages adopts either a Rp or a Sp configuration. Stereopure Rp-(PS-ODNs)have been prepared enzymatically and exhibited a lower stability to nucleases endogenous to human serum than the parent PS-ODNs with undefined P-chirality. Conversely, chemically synthesized stereopure Sp-(PS-ODNs) have demonstrated superior stability to these nucleases than P-diastereomeric PS-ODNs. Thus, to further investigate the biological, pharmacokinetic, and toxicologic properties of P-stereopure PS-ODNs improved chemical methods are required to synthesize these biomolecules and increase their availability for clinical studies. We have discovered that deoxyribonucleoside cyclic N-acylphosphoramidites are efficient monomers for the stereospecific synthesis of PS-ODNs. Indeed, base-assisted condensation of the 5'-OH function of a nucleoside or a nucleotide covalently linked to a solid support with deoxyribonucleoside cyclic N-acylphosphoramidites led to rapid and efficient formation of an internucleoside phosphite triester linkage. The phosphite triester function can be either oxidized to a phosphate triester(PO) by treatment with tert-butyl hydroperoxide or sulfurized by a sulfur transfer reagent to a P-stereodefined thiophosphate phosphotriester(PS)function to permit, for example, stereocontrolled synthesis of chimeric PO/PS-ODNs. In this context, the stereospecific synthesis of the PS-ODNs [Rp,Rp]- and [Sp,Sp]-d(CpsCpsC), [Rp,Sp,Rp]-d(CpsCpsCpsC), and [Rp]11- d(Tps)11T have been successfully accomplished. The method is currently being optimized through the use of new deoxyribonucleoside cyclic N-acylphosphoramidite monomers and improved solid supports to enable incorporation of the four different nucleobases into DNA chains of at least 20 bases long. Incidentally, a novel Nuclear Magnetic Resonance method has been developed to permit rapid determination of the absolute configuration of deoxyribonucleoside cyclic N-acylphosphoramidites at phosphorus. This methods further confirms the P-stereochemistry of any phosphorothioated oligonucleotides synthesized from these phosphoramidites. During the course of our investigation, we have discovered that phosphate/thiophosphate protection when using cylic N-acyl phosphoramidites is heat-sensitive. This discovery led to the development of a number of novel thermolabile phosphate protecting groups. These include the 2-(N-formyl-N-methyl)aminoethyl, 4-oxopentyl, and the 3-[(N-tert-butyl)carboxamido]-1-propyl groups. We have also recently found that these unique phosphate protecting groups could also be adapted to the 5'-OH protection of nucleoside and oligonucleotides. In this regard, sulfur-containing groups for 5'-OH protection of nucleosides and oligonucleotides have been identified and are currently being investigated for potential application to the synthesis of oligonucleotides on arrays. These findings along with the fact that deoxyribonucleoside cyclic N-acylphosphoramidites can, unlike conventional deoxyribonucleoside phosphoramidites, effectively produce oligonucleotides under relatively wet conditions are attractive features for parallel synthesis of oligonucleotides on arrayable surfaces. Oligonucleotide microarrays can be invaluable for analyzing gene expression from, for example, tumorigenic versus non-tumorigenic human cell lines, and high-throughput screening of point mutations and single nucleotide polymorphisms in genomic DNA that predispose human to diseases.